This application addresses broad Challenge Area (06) Enabling Technologies, and specific Challenge Topic 06-CA-102: Transient Molecular Complexes in Cancer. Virtually all cellular processes, including those involved in cell growth, differentiation, and apoptosis, rely on specific and carefully regulated protein-protein association and dissociation reactions. Dysregulation of these reactions is characteristic and frequently a cause of many human cancers. Because the interactions that underlie protein complexes are often weak and transient, the identification and characterization of critical complexes often has been extremely difficult or unsuccessful. Nowhere has this problem been more evident than with ubiquitin conjugation (E3 ubiquitin ligase) and deubiquitination (DUB) enzymes. Covalent modification of proteins by one or more molecules of the 76-amino acid ubiquitin is the major route of regulated intracellular proteolysis in all eukaryotes, and as such it is responsible for the control of numerous key regulatory proteins. Additionally, depending upon the specific mono or polyubiquitin modification, ubiquitination also can lead to other fates. Thus, ubiquitin signals are used in endocytosis and protein trafficking, transcription activation, kinase activation cascades, and chromatin remodeling. Of the many hundreds of different E3 and DUB enzymes expressed in human cells, physiological substrates are known for only a handful. The transient nature and low-abundance of these enzyme.substrate complexes are among the principal reasons that most attempts to identify substrates by two- hybrid assays or affinity-based pulldown experiments have failed. The goal of this proposal is to overcome these limitations with a new approach termed cognate-complex biotin tagging ("CBiT"). In Aim 1, modified biotin ligase and biotin acceptor peptide constructs will be designed to allow biotinylation only when these two components are brought together by interaction of an enzyme (E3 or DUB) with its substrate. By having the biotin acceptor peptide fused to ubiquitin, even transient E.S complexes in vivo will be subject to biotinylation, and only ubiquitin conjugates will be tagged. As a result, bona fide substrates should be highly enriched after affinity-isolation of biotinylated proteins and amenable to identification by mass spectrometry. The CBiT strategy will be tested in vivo in yeast with known E3.substrate and DUB.substrate pairs. In Aim 2, cell lines and DNA vectors to implement CBiT will be optimized for efficient application with human E3s and DUBs;several E3s and DUBs that have been implicated in human cancers and other diseases will be used in tests of the CBiT approach. PUBLIC HEALTH RELEVANCE: Aberrant formation or stability of protein complexes is characteristic of many human cancers and other diseases. Because the interactions that underlie protein complexes are often weak and transient, attempts to identify and characterize critical protein interactions frequently are unsuccessful or extremely difficult. To overcome this problem, a new approach termed cognate-complex biotin tagging ("CBiT") will be developed and applied to complexes that regulate ubiquitin-dependent signaling.